This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycosylation is one of most important and widespread types of post-translational modifications of proteins. Glycosylation patterns may be altered by physiological changes and disease, and have shown some promise as biomarkers for early cancer detection. Glycosylated proteins, however, pose difficult analytical challenges, and hence the role and function of glycosylation, both in healthy and diseased organisms, has remained rather mysterious. One of the difficulties is the remarkable heterogeneity of glycoproteins: in some proteins, glycosylation sites may feature a wide array of different oligosaccharides, while other molecules of the same protein bear no sugars at all at the same site. Hence there is a great need for improved analytical techniques for studying glycoproteins.We have been developing enrichment methods for both secreted O-linked glycopeptides and intracellular O-GlcNAc modified molecules. We are able to test the success of enrichment strategies in-house using LC/MS/MS analysis on our QTOF instrument, however glycopeptide identification and especially modification site-assignments require electron-transfer dissociation analysis.